freezing artifact..muscle bx's

From:Sharon Allen <>

In our lab we do approx. 10 to 15 muscle bx's per month and have pretty well
eliminated the problem of freezing artifact, by using a thermometer and not
emerging the muscle into the isopentane until the temp. has reached -150oC.
Before we purchased the thermometer, we guessed at the temp. and had some
Freezing artifact can also be caused by the specimen being to wet. Have it
sent in saline moistened gauze, not floating in saline (as the OR's seem to
like to do). If it comes wet, blot it almost dry before freezing.
We use liquid nitrogen and isopentane for freezing and have the muscle taken
in muscle clamps.
1.  Suspend bullet in the liquid nitrogen to cool.
2.  Suspend a beaker containing 40-50 ml. of isopentane in the liquid
nitrogen, placing the thermometer in the beaker.
3.  While the isopentane is cooling, we place a small amount of gum
tragacanth on a piece of cork(works great, floats upside down).
4.  Cut one clamped end off the muscle and embed it upright in the gum
trag., having only the other clamped end touching the gum trag.
5.  When temp. has reached -150oC, drop the specimen in the isopentane,
keeping the muscle straight.  Allow it to freeze no longer than a minute (to
long can cause fracturing of the tissue).
6.  We then put the frozen muscle into a -70oC freezer for an hour or two
before cutting.  We have found it cuts much better if it is left to
stabalize (no explanation for this).  
7.  When ready to cut the muscle, we let it sit for 20 to 30 min. in the
cryostat before cutting to bring it up to -20oC.  We use O.C.T. compound to
attach the cork base to the cryostat chuck.
Hope this will be of some help to you.

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